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AIM: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. MATERIALS AND METHODS: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. RESULTS: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. CONCLUSION: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

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The aim of the study was to develop a new technology for the detection of amyloid in human tissues based on the fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo-2-naphthylazo)fluorene (DSNAF). MATERIALS AND METHODS: Synthesis of DSNAF was performed by diazotization of 2,7-diaminofluorene in a stream of argon followed by azo coupling with naphthionic acid. Identification of DSNAF was performed using MALDI mass spectrometry. Human myocardial samples from males and females aged from 85 to 98 years (n=11) were the material for the histochemical study. Myocardial paraffin sections were stained with a 0.1% aqueous solution of Congo red or with an aqueous solution (0.1 or 0.034%) of DSNAF under the same conditions. RESULTS: It has been demonstrated for the first time that a new fluorene-based analogue of Congo red, DSNAF, can be successfully used to identify amyloid deposits in histological sections of human myocardium. In terms of the specificity and intensity of amyloid staining, DSNAF is comparable to Congo red, which is the gold standard for detecting amyloid deposits. The fluorescence intensity of DSNAF when binding to amyloid fibrils is significantly higher than the intensity of Congo red fluorescence (with a lower intensity of background fluorescence of heart muscle tissue). This is especially useful for identifying small deposits of amyloid in the human tissues which is important when using small biopsies. CONCLUSION: The advantages of using DSNAF allow us to consider the developed technology for the detection of amyloid as a new promising method of identifying amyloid deposits in human tissues.

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By means of spectrophotometric assay we investigated interaction of the dye Congo red (CR) with fibrils of model proteins--hen egg white lysozyme, recombinant human beta2-microglobulin (b2M) and recombinant human transthyretin (TTR). The commercial dye sample was found to contain a significant amount of impurities. Methods for the dye purification are disclosed and CR molar extinction coefficient at 490 nm (ε490) was determined to be 3.3 x 10(4) M(-1) x cm(-1) at pH above 6.0. Formation of the CR-fibril complex results in changes in the dye visible absorption spectrum. According to the data on titration of fibril solutions with excess of the dye, CR binds to lysozyme fibrils at a ratio of about 5 molecules per protein monomer within fibril structure, to b2M fibrils--about 4 molecules per monomer, to TTR fibrils--about 4 molecules per subunit of the protein.

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The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

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The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and ß2-microglobulin (ß2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and ß2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and ß2M that are suitable for further studies.

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Polypeptide chain fragments of recombinant transthyretin (TTR) with leucine-55 substituted by proline (L55P), which are involved in abnormal fibrillogenesis of this protein, were studied. No fibrils were produced in purified preparations of TTR(L55P) under the optimum conditions for fibrillogenesis but in absence of protease inhibitors. The ability of TTR for fibrillogenesis was lost because of a limited proteolysis resulting in detachment of the TTR polypeptide chain C-terminal fragment of approximately 18 amino acid residues in length. This proteolysis seemed to occur with involvement of a bacterial serine endopeptidase sohB (EC 3.4.21), which was identified in TTR preparations by the MALDI-TOF method. The presence of the C-terminal fragment of the TTR polypeptide chain seems to be crucial for production of abnormal fibrils.

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